Human Reproduction, Vol.28, No.3 pp. 740–749, 2013
Advanced Access publication on December 19, 2012 doi:10.1093/humrep/des432
ORIGINAL ARTICLE Reproductive biology
Human trophectoderm cells are not
yet committed
Caroline De Paepe1,†, Greet Cauffman1,2,†, An Verloes3,
Johan Sterckx2, Paul Devroey2, Herman Tournaye2,
Inge Liebaers1,4, and Hilde Van de Velde1,2,*
1
Department of Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels 1090, Belgium 2Centre for
Reproductive Medicine (CRM), Universitair Ziekenhuis Brussel (UZ Brussel), Laarbeeklaan 101, Brussels 1090, Belgium 3Department of
Hematology, Universitair Ziekenhuis Brussel (UZ Brussel), Laarbeeklaan 101, Brussels 1090, Belgium 4Centre for Medical Genetics (CMG),
Universitair Ziekenhuis Brussel (UZ Brussel), Laarbeeklaan 101, Brussels 1090, Belgium
*Correspondence address. Tel: +3224776696; Fax: +3224776692; E-mail: [email protected]
Submitted on September 7, 2012; resubmitted on October 18, 2012; accepted on November 8, 2012
study question: Are human trophectoderm (TE) cells committed or still able to develop into inner cell mass (ICM) cells?
summary answer: Human full blastocyst TE cells still have the capacity to develop into ICM cells expressing the pluripotency marker
NANOG, thus they are not yet committed.
what is known already: Human Day 5 full blastocyst TE cells express the pluripotency markers POU5F1, SOX2 and SALL4
as well as the TE markers HLA-G and KRT18 but not yet CDX2, therefore their developmental direction may not yet be definite.
study design, size, duration: The potency of human blastocyst TE cells was investigated by determining their in vitro capacity
to develop into a blastocyst with ICM cells expressing NANOG; TE cells were isolated either by aspiration under visual control or after
labeling with fluorescent 594-wheat germ agglutinin. Further on, aspirated TE cells were also labeled with fluorescent PKH67 and repositioned in the center of the original embryo.
participants/materials, setting, methods: Human preimplantation embryos were used for research after obtaining
informed consent from IVF patients. The experiments were approved by the Local Ethical Committee and the ‘Belgian Federal Committee
on medical and scientific research on embryos in vitro’. Outer cells were isolated and reaggregated by micromanipulation. Reconstituted
embryos were analyzed by immunocytochemistry.
main results and the role of chance: Isolated and reaggregated TE cells from full human blastocysts are able to develop
into blastocysts with ICM cells expressing the pluripotency marker NANOG. Moreover, the majority of the isolated TE cells which were
repositioned in the center of the embryo do not sort back to their original position but integrate within the ICM and start to express
NANOG.
limitations, reasons for caution: Owing to legal and ethical restrictions, manipulated human embryos cannot be transferred into the uterus to determine their totipotent capacity. The definitive demonstration that embryos reconstructed with TE cells are
a source of pluripotent cells is to obtain human embryonic stem cell ‘like’ line(s), which will allow full characterization of the cells.
wider implications of the findings: Our finding has important implications in reproductive medicine and stem cell biology
because TE cells have a greater developmental potential than assumed previously.
study funding/competing interest(s): Scientific Research Foundation—Flanders (FWO-Vlaanderen) and Research
Council (OZR) of the Vrije Universiteit Brussel. None of the authors declared a conflict of interest.
Key words: trophectoderm / inner cell mass / human blastocyst / NANOG / plasticity
†
These authors contributed equally to the work.
& The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
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Human trophectoderm is not yet committed
Introduction
Preimplantation development starts with the fusion of two highly differentiated cells—the oocyte and the spermatozoon—resulting in a
totipotent zygote. Totipotency of a single cell refers to its ability to
generate offspring or to contribute to all embryonic and extraembryonic tissues (Adjaye et al., 2008; Tarkowski et al., 2010). The differentiation capacity of early embryonic cells (blastomeres) is subject to
continuous investigation. In animal models, the totipotency of blastomeres can be demonstrated by transferring embryos derived from a
single blastomere into a pseudo-pregnant foster mother in order to
obtain offspring (Tarkowski et al., 1967; Johnson et al., 1995). Experiments have shown that the potential of blastomeres depends on the
cell stage of the embryo from which they are derived, the position
of the blastomeres (inner versus outer) and their cellular mass (Tarkowski et al., 2001; Suwinska et al., 2008).
Owing to both ethical and legal restrictions, early human development can only be studied in vitro. The capacity of a single blastomere
to develop into a blastocyst with an inner cell mass (ICM) and trophectoderm (TE) cells or its ability to contribute to both the ICM and
the TE demonstrates at least pluripotency. Only limited data exist
on the potency of human blastomeres and it remains unknown at
which point of time their totipotent capacity is lost. Recently, we
showed that all four blastomeres of a 4-cell stage embryo are able
to develop individually into blastocysts with an ICM and TE cells
(Van de Velde et al., 2008). The birth of a child after transferring a
Day 2 embryo of which only one out of four cells had survived the
freezing-thawing procedure confirmed that at least one of the 4-cell
stage blastomeres is totipotent (Veiga et al., 1987). Furthermore, it
was shown that human embryonic stem cell (hESC) lines can be
derived from both single 4-cell stage (Feki et al., 2008; Geens et al.,
2009) and 8-cell stage (Klimanskaya et al., 2006; Klimanskaya et al.,
2007) blastomeres and blastomeres from early arrested embryos
(Zhang et al., 2006; Lerou et al., 2008), indicating that some of the
early blastomeres are at least pluripotent.
The aim of this study was to investigate the potency of outer cells
from the compaction stage and the blastocyst stage in the human.
We hypothesized that the outer cells from compacted embryos,
early and full blastocysts (blastocyst scoring according to Gardner
and Schoolcraft, 1999) still have the capacity to develop into ICM
cells because they express the pluripotency markers POU5F1,
SOX2 and SALL4 (Cauffman et al., 2006; Cauffman et al., 2009;
Chen et al., 2009) as well as the TE markers HLA-G (Verloes
et al., 2011) and KRT18 (Cauffman et al., 2009) but not yet CDX2
(Chen et al., 2009).
Materials and Methods
The experiments were approved by the Local Ethical Committee and the
‘Belgian Federal Committee on medical and scientific research on embryos
in vitro’. Compacted embryos (14– 28 cells, no fragmentation) and top
quality full and expanding blastocysts (≥32 cells, Bl3AA and Bl4AA,
respectively, blastocyst scoring according to Gardner and Schoolcraft
(1999)), which were diagnosed to carry a genetic disease after removal
of one blastomere on Day 3, were used after obtaining informed
consent. For some experiments, cryopreserved 8-cell stage embryos
were thawed and cultured to the blastocyst stage, after becoming available
for research after the legally determined 5-year period of storage, and if
consent was previously obtained.
Micromanipulation and culture
Embryos were biopsied in Ca2+Mg2+-free medium (Vitrolife, Stockholm,
Sweden) using laser biopsy. Blastocysts were decavitated by aspirating
the fluid out of the blastocoele using an ICSI pipette. Decavitated blastocysts and compacted embryos were incubated in Ca2+Mg2+-free medium
until full decompaction. Blastomeres were manipulated using a self-made
pipette with an inner diameter of 40 mm. Outer blastomeres were aspirated under visual control. Several steps were undertaken to exclude contamination with inner cells: the hole for biopsy was larger than usual in
order to be able to intrude the pipette into the perivitelline space
without disturbing the position of the blastomeres; by orientating and
firmly fixing the blastocyst at the embryonic pole, exclusively mural TE
blastomeres were removed; the pipette was never inserted inside the
embryo and blastomeres were only taken when they were immediately accessible at the laser hole; blastomeres were taken in one move in order
not to re-intrude the pipette and disturb the position of the blastomeres;
aspiration was stopped whenever there was the smallest doubt on the original position of the blastomere. All steps were carried out under visual
control of two persons. Embryos were reconstituted by putting the blastomeres in an empty zona pellucida. Reconstituted ‘outer-cells-only’
embryos and the founder (remaining outer + all inner cells) embryos
were cultured individually in blastocyst medium (Vitrolife, Stockholm,
Sweden; Sage, Pasadena, CA, USA) in 25 ml droplets under oil in 5%
O2 6% CO2 conditions up to Days 6 and 7 of preimplantation development. For each experiment, unmanipulated embryos were cultured as
well. The newly formed blastocysts were morphologically scored before
fixation according to Gardner and Schoolcraft (1999): a good-quality
blastocyst was considered to be at least Bl3BB. For each experiment, nonmanipulated embryos were cultured, serving as controls (data not shown).
Immunocytochemistry
Embryos were analyzed by immunocytochemistry and confocal microscopy for the expression of NANOG (ICM) and HLA-G (TE) as described
(Verloes et al., 2011). Embryos were fixed for 10 min with 3.7% formaldehyde (Merck, VWR International), permeabilized for 20 min with 0.1%
Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) at room temperature
and incubated overnight at 48C with the mouse monoclonal immunoglobulin (Ig) G1 anti-HLA-G antibody MEM-G/9 (8 mg/ml; Santa Cruz
Biotechnology, CA, USA), a mouse monoclonal IgG1 anti-NANOG
antibody (5 mg/ml; Abcam, Cambridge, UK) and/or rabbit polyclonal antiNANOG antibodies (3 mg/ml; Abcam). Control reactions for non-specific
binding were included in each experiment by replacing the antibodies with
a mouse monoclonal IgG1 antibody (BD Biosciences, Erembodegem,
Belgium) or rabbit IgGs (Abcam) at the same concentration as the
primary antibodies. Alexa Fluor 647-conjugated goat anti-mouse F(ab’)2
fragments (Molecular Probes, Invitrogen, Gent, Belgium), Alexa Fluor
488-conjugated donkey anti-mouse IgG (Molecular Probes, Invitrogen)
and Alexa Fluor 488-conjugated donkey anti-mouse IgGs (Molecular
Probes, Invitrogen) were used as secondary antibodies at a concentration
of 10 mg/ml for 2 h at 48C in the dark. All primary and secondary antibody solutions were prepared in phosphate-buffered saline supplemented
with 2% bovine serum albumin (Sigma-Aldrich). After staining, embryos
were mounted between two glass cover slips (24 × 50 mm) in the SlowFadew Gold antifade reagent and round glass cover slips (diameter of
10 mm) were put between the cover slips to prevent squeezing. Confocal
scanning microscopy with an Ar-HeNe laser (488/633) (IX71 Fluoview
300; Olympus, Aartselaar, Belgium) was performed to record the fluorescent images.
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Blastomere labeling and tracing
To separate outer from inner cells, blastocysts were labeled with Alexa
Fluor 594-wheat germ agglutinin (594-WGA) (Molecular Probes, Invitrogen) (Wright, 1984; Basbaum and Menetrey, 1987; Hoshino et al.,
2010). Blastocysts were incubated for 10 min in 1 mg/ml pronase
(Sigma-Aldrich) to remove the zona pellucida, rinsed in the HEPES
medium (Gynotec, Malden, The Netherlands) and cultured for 30 min in
blastocyst medium to recover before labeling. Subsequently, they were
incubated for 10 min in 5 mg/ml 594-WGA and finally rinsed in the
HEPES medium; blastocysts that collapsed during the procedure were
not used further. Blastocysts were decavitated as described above and dissociated in Ca2+Mg2+-free medium by pipetting. Blastomeres were put individually in 2 ml droplets of HEPES medium and labeling was evaluated
using an Axioplan 2 Imaging HBO100 microscope (Zeiss, Zaventem,
Belgium). Labeled and unlabeled blastomeres were separated, collected
in one droplet of HEPES medium and embryos were reconstituted by
putting separated blastomeres in an empty zona pellucida using a 40 mm
biopsy pipette.
For tracing experiments, outer blastomeres were isolated from blastocysts, as described above by micromanipulation under visual control of
two persons and labeled using PKH67 Fluorescent Cell Linker kit
De Paepe et al.
(Sigma-Aldrich) (Tarkowski et al., 2010). Blastomeres were individually
incubated for 30 s in 1/200 dye solution while pipetting. They were
rinsed in Ca2+Mg2+-free medium and placed in the center of the
founder embryo using a 40 mm biopsy pipette.
Results
We performed three types of manipulations to determine at which
stage the decision of a blastomere to become either an ICM or a
TE cell is irreversibly taken (Fig. 1).
First (manipulation 1), we isolated the outer cells by aspiration
under visual control (Fig. 1A). We investigated the potency of outer
cells from embryos at the compaction stage (Day 4) and at the blastocyst stage (Days 5 and 6) to develop in vitro into blastocysts with both
ICM and TE cells. (Table I and Fig. 2). The vitality of the de novo
formed blastocysts was determined by both morphological scoring
(Gardner and Schoolcraft, 1999) and immunocytochemistry determining the integrity of the TE epithelium (HLA-G expression) (Verloes
et al., 2011) and the potency of the ICM cells (NANOG expression)
(Mitsui et al., 2003; Hyslop et al., 2005; Cauffman et al., 2009) (Fig. 3).
Figure 1 Schematic representation of the manipulations. (A) Human blastocysts were decavitated and decompacted, outer blastomeres of two
blastocysts were aspirated under visual control, embryos were reconstituted by putting the outer blastomeres in an empty zona pellucida, reconstituted embryos were cultured up to Days 6 and 7 of preimplantation development and immunostained for NANOG (red) and HLA-G (purple);
(B) the zona pellucida of the blastocysts was removed, blastocysts were labeled with 594-wheat germ agglutinin (WGA) (yellow), decavitated and
decompacted, labeled and unlabeled blastomeres were separated by immunofluorescence microscopy, labeled outer cells were collected and put
in an empty zona pellucida, reconstituted embryos were cultured up to Days 6 and 7 of preimplantation development and immunostained for
NANOG (red); (C) blastocysts were decavitated and decompacted, outer blastomeres were aspirated under visual control, labeled with PKH67
(green) and placed in the center of the founder embryo, the position of the labeled cells was determined on Day 6 of preimplantation development,
the embryos were immunostained for NANOG (red). The number of blastomeres drawn and the colors used are not representative of the manipulations performed.
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Human trophectoderm is not yet committed
Table I Human TE cells are not yet committed.
Experiment
Stage
Embryos
manipulated (n)
Blastocysts
developed (n)
Blastocysts which
developed an ICM (n)
.............................................................................................................................................................................................
1
2
3
4
5
Compaction Day 4
1 founder
Outer-cells-only (5– 9 cells)
11
11
10
8
8
0
Compaction Day 4
2 founder
Outer-cells-only (12– 14 cells)
22
11
22
11
17
10
Bl1 –Bl2 Day 5
2 founder
Outer-cells-only (10– 23 cells)
10
5
9
3
8
2
Bl3 Day 5
2 founder
Outer-cells-only (19– 30 cells)
6
3
5
2
5
1
Bl3 Day 5
3 founder
Outer-cells-only (21– 45 cells)
6
2
6
2
6
2
Outer cells were extracted from one (experiment 1), two (experiments 2, 3 and 4) or three (experiment 5) embryos at different stages and days of preimplantation development:
compaction on Day 4 (experiments 1 and 2); early blastocysts Bl1 and Bl2 (experiment 3) on Day 5 and full blastocyst Bl3 on Day 5 (experiments 4 and 5). The founder embryos
and the ‘outer-cells-only’ embryos were cultured until Days 6 and 7, developing into blastocysts with a clearly visible ICM.
First we investigated the potency of outer cells from compacted
embryos. A limited number of outer cells (5–9) was aspirated and
reaggregated in an empty zona pellucida. The ‘outer-cells-only’
embryos recompacted and developed into blastocysts but none of
them appeared to have an ICM (Table I, Figs 2A and 3A). This
could be due to loss of capacity at compaction or to insufficient cell
mass to generate an inner population. To compensate for the presumed insufficient cell mass, we reaggregated outer cells from two distinct embryos (12 –14 cells in total). These ‘outer-cells-only’ embryos
recompacted and developed into good-quality blastocysts with an ICM
in which NANOG was found (Table I, Figs 2B and 3B). Next we investigated the potency of outer cells from early blastocysts. Outer cells
from two blastocysts (10 –23 cells in total) were reaggregated. They
recompacted, recavitated and some developed into blastocysts with
ICM cells expressing NANOG (Table I). In case of full blastocysts,
we reconstituted embryos with outer cells from two blastocysts
(Table I, Figs 2C and 3C) and three blastocysts (Table I and
Fig. 3D) to have sufficient cellular mass (19 –45 cells in total). The
‘outer-cells-only’ embryos recompacted, recavitated and developed
into blastocysts with an ICM expressing NANOG. The majority of
the founder embryos developed into good-quality blastocysts with
an ICM in which NANOG was found (Table I). Similar experiments
with Days 5 and 6 expanded blastocysts failed (data not shown).
Day 5 expanding blastocysts could be decavitated and decompacted;
however, isolated TE cells used to reconstitute embryos were not able
to recompact but vacuolated. Day 6 expanded blastocysts could not
be completely decavitated and decompaction did not occur. Additional attempts at Days 5 and 6 to isolate TE cells by laser cutting (Schoolcraft et al., 2010) and incubation in Ca2+Mg2+-free medium to obtain
single TE cells resulted in significant cell death. Our results show that
outer cells from human full blastocysts can still change their developmental direction, whereas outer cells from expanding blastocysts no
longer have this capacity.
To confirm our results (manipulation 2), we performed additional labeling experiments in which we could use nearly all TE cells from the
original blastocyst and thus we did not need to pool outer cells from distinct embryos (Fig. 1B). Outer cells were labeled with 594-WGA. To
test the specificity of 594-WGA, we labeled one compacted embryo
and four full blastocysts; 594-WGA was shown to stick to the membrane of the outer cells and not to enter the cavity; labeled inner cells
were not detected (Fig. 4A). We separated labeled outer and unlabeled
inner cells from six full blastocysts by fluorescence microscopy and
reconstituted ‘outer-cells-only’ embryos with labeled cells. The in vitro
development of the reconstituted embryos was delayed because
recompaction after 594-WGA labeling seemed to be impaired. Three
out of six ‘outer-cells-only’ embryos developed into good-quality blastocysts with an ICM on Days 6 and 7 (Table II and Fig. 4B). From one of
the six full blastocysts we also reconstituted an embryo with unlabeled
‘inner-cells-only’, it developed into a good-quality blastocyst with an
ICM on Day 7. NANOG was found in ICM cells of both the
‘inner-cells-only’ and ‘outer-cells-only’ embryo on Day 7, supporting
our hypothesis that outer cells of a full human blastocyst have the capacity to change their developmental direction as well as inner cells.
Next we hypothesized that the outer cells can change their developmental direction when they are put in an inner position. To investigate this (manipulation 3), we repositioned labeled TE cells from a full
human blastocyst in an inner position (Fig. 1C). However, 594-WGA
could not be used to trace outer cells because it disappeared from the
membrane after 16 h in vitro culture. Therefore a limited number of
outer cells (maximum eight to avoid contamination with inner cells)
were extracted and labeled with PKH67 Fluorescent Cell Linker kit
(Tarkowski et al., 2010). This lipid dye could be used for cell tracing
after 24 h in vitro culture but not after 48 h. Labeled cells were
placed in the center of the decavitated and decompacted founder
embryo and their position was determined after 20 –24 h in vitro
culture using confocal microscopy. The manipulated embryos
744
De Paepe et al.
Figure 2 ‘Outer-cells-only’ human embryos can develop into a blastocyst with ICM cells. (A) Decompacted embryo (A1) on Day 4,
‘outer-cells-only’ (A2) developed further into blastocysts without an ICM (A4), whereas the founder embryo developed into a blastocyst with an
ICM (A3) on Day 6; (B) two decompacted embryos (B1 and B2), ‘outer-cells-only’ (B3) developed into blastocysts with an ICM (B6) as well as
the founder embryos (B4 and B5) on Day 6; (C) two full blastocysts (C1 and C2) on Day 5, ‘outer-cells-only’ (C3) developed into blastocysts
with an ICM (C6) as well as the founder embryos (C4 and C5) on Day 6. The arrow indicates the ICM. Images taken using an inverted microscope:
Nikon Eclipse TE300.
developed into blastocysts with an ICM (Table II). We detected the
majority of the PKH67-labeled cells in the ICM of the newly formed
blastocysts (88.7%) (Fig. 5), indicating that the outer cells integrated
within the developing ICM of the founder embryo. In these ICM,
NANOG was found in PKH67-labeled cells (54.7%) as well as in
PKH67-unlabeled founder cells. We conclude that the TE cells
from a full human blastocyst have the capacity to change their developmental direction when put in an inner position, rather than sorting
back to their original position.
Discussion
We found that TE cells from a full human blastocyst as well as ICM
cells have the capacity to develop in vitro into a blastocyst with ICM
cells expressing NANOG and we conclude that these cells can still
change lineage direction. We suggest that human full blastocyst TE
cells as well as ICM cells are potentially totipotent. Their totipotent
capacity will never be proven because they cannot be transferred
into a uterus. When put in an inner position, full blastocyst TE cells
can integrate within the ICM and start to express NANOG, indicating
that their lineage direction is determined by position. Since full blastocyst TE cells still express essential master genes of pluripotency (Cauffman et al., 2006; Cauffman et al., 2009; Chen et al., 2009) as well as
epithelium markers (Cauffman et al., 2009; Verloes et al., 2011), they
probably do not need to completely alter their expression profile. The
lineage direction of TE cells is irreversible at the expanding blastocyst
stage and corresponds with the onset of nuclear CDX2 expression
(Chen et al., 2009) and the down-regulation of nuclear expression
of the pluripotency key players SOX2 (Cauffman et al., 2009) and
POU5F1 (Chen et al., 2009).
Our primary aim was to investigate the potency of outer cells
from full blastocysts because they still express pluripotency
markers. Therefore we decavitated and decompacted the blastocysts and aspirated single TE cells as for cleavage-stage biopsy.
The cells of early and full blastocysts did not degenerate after decavitation, decompaction and aspiration, and they recompacted
Human trophectoderm is not yet committed
745
Figure 3 ‘Outer-cells-only’ human embryos can develop into a blastocyst with ICM cells expressing the pluripotency marker NANOG. NANOG
(green in 2 and 3) and HLA-G (red in 3) immunocytochemistry in Day 6 blastocysts reconstituted with outer-cells-only from (A) one Day 4 compacted embryo; (B) two Day 4 compacted embryos; (C) two Day 5 full blastocysts; (D) three Day 5 full blastocysts; and (E) a founder full blastocyst.
The phase contrasts of the embryos are shown in (1).
easily. They developed into blastocysts with a cohesive TE expressing HLA-G (Verloes et al., 2011) and an ICM. NANOG was found
in some but not all ICM cells, as described (Mitsui et al., 2003;
Hyslop et al., 2005; Cauffman et al., 2009). We only took a
limited number of outer cells from two to three blastocysts in
order not to have contamination with inner cells on the one hand
and to have enough cells to have an inner population in the newly
formed embryos on the other hand. It was not possible to reconstitute embryos with the original number of blastomeres because of
the scarcity of top quality embryos at the same developmental
stage available for research.
To confirm our findings, we did additional experiments in which
outer cells were labeled prior to biopsy. Experiments performed to
specifically label outer cells with PKH67 Fluorescent Cell Linker,
as described in the mouse (Suwinska et al., 2008), failed. In all experimental conditions tested (incubation time and dye concentration),
outer and inner cells from compacted embryos and blastocysts were
labeled. The distinct labeling results obtained in the mouse and the
human are most likely related to differences in the membrane of the
blastomeres between the two species We were able to label outer
cells with 594-WGA which sticks to the membrane of the outer
cells and could be used to separate them from the inner cells by
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De Paepe et al.
Figure 4 TE cells as well as ICM cells from a human full blastocyst are not yet committed. A Day 5 blastocyst was labeled with 594-WGA (A1);
labeled outer cells and unlabeled inner cells could be distinguished by immunofluorescence microscopy (A2) and separated one-by-one into labeled
(A3) and unlabeled (A4) cells; an embryo reconstituted with outer-cells-only (B2) developed into a blastocyst with ICM cells (phase contrast in B4)
expressing NANOG (green in B6); an embryo reconstituted with inner-cells-only (B3) developed into a blastocyst with ICM cells (B3; phase contrast
in B5) expressing NANOG (green in B7). The arrows indicate the ICM. The original embryo is shown in (B1).
747
Human trophectoderm is not yet committed
Table II Human full blastocyst TE cells are not yet committed.
Experiment
Stage
Embryos
reconstituted (n)
Blastocysts
developed (n)
Blastocysts which
developed an ICM (n)
.............................................................................................................................................................................................
1
2
Bl3 Day 5
Outer-cells-only (20 –36 cells)
Inner-cells-only (25 cells)
6
1
3
1
3
1
Bl3 Day 5
6
5
5
(Experiment 1) outer cells were labeled with 594-wheat germ agglutinin to separate outer from inner cells, embryos were reconstituted with labeled ‘outer-cells-only’ or unlabeled
‘inner-cells-only’ and cultured further until Days 6 and 7; (Experiment 2) four to eight outer cells extracted from Day 5 full blastocysts Bl3 were labeled with PKH67, repositioned in the
center of the founder decavitated and decompacted embryos, and the embryos were cultured further until Day 6.
Figure 5 Human TE cells put in an inner position do not sort back to their original position and start to express NANOG. Isolated outer cells from
a Day 5 full blastocyst were labeled with the PKH67 green fluorescent dye and reintroduced in the center of the founder decavitated and decompacted
embryo. The labeled outer cells integrated within the founder ICM (green in 2) and expressed the pluripotency marker NANOG (red in 3) on Day
6. The green/red combination is shown in (4), the arrows indicate PKH67-labeled cells expressing NANOG. The phase contrast of the reconstituted
embryo is shown in (1).
fluorescence microscopy. We confirmed that ‘outer-cells-only’ of a full
human blastocyst can develop into blastocysts with an ICM, as well as
‘inner-cells-only’. However, 594-WGA labeling interfered with recompaction and resulted in a developmental delay. Therefore, NANOG
was not found in Day 6 ICM cells as described (Mitsui et al., 2003;
Hyslop et al., 2005; Cauffman et al., 2009) but only in some Day 7
ICM cells.
We also found that human full blastocyst TE cells are capable of
changing lineage direction if they are put in an inner position, indicating
that position determines fate (Tarkowski, 1967). These TE cells could
integrate within the ICM and some cells started to express NANOG,
confirming that they can become precursor epiblast cells (Mitsui et al.,
2003; Hyslop et al., 2005; Cauffman et al., 2009). Tracing outer cells
with 594-WGA could not be performed because it disappeared
after 16 h in vitro culture (Day 6) and NANOG could only be detected
on Day 7. Although PKH67 could not be used to specifically label
outer cells in the blastocyst, we could use PKH67 to label the outer
cells after isolation. Also, only a limited number of outer cells were
extracted in order to exclude contamination with inner cells. The majority of the labeled and repositioned outer cells did not sort back to
an outer position but integrated within the ICM and started to express
NANOG, confirming that they can change lineage direction.
The outer cells of expanding Day 5 human blastocysts could be isolated but the single cells were unable to recompact, and vacuolated;
the outer cells of an expanded Day 6 blastocyst formed a strong epithelium and the cells could no longer be disaggregated into single cells,
suggesting that at these stages the TE cells have lost their plasticity to
change developmental direction. This stage of development (i.e.
expansion) coincides with the presence of CDX2 and the downregulation of SOX2 and POU5F1 transcription factors in the nuclei
of TE cells (Cauffman et al., 2009; Chen et al., 2009).
Similar experiments have been performed in the mouse and cattle,
however, with a different outcome. Embryos reconstituted with inner
or outer cells from compacted mouse embryos (16 cells) to the original cell number can develop into offspring, indicating that inner
cells as well as outer cells are totipotent (Suwinska et al., 2008).
The inner cells of early blastocysts (32 cells) are able to develop
into new blastocysts with an ICM but they fail to implant, indicating
that they are pluripotent but not totipotent (Suwinska et al., 2008).
On the other hand, the outer cells of early blastocysts recavitate
but fail to develop an ICM, indicating loss of totipotency and pluripotency. We believe this is related to their inability to recompact properly and generate an inner cell population after manipulation. Recently,
using tetraploid carrier blastomeres able to form only TE cells, it was
shown that single outer cells from a mouse 32-cell stage embryo
can sporadically develop into a fetus (1/42 embryos transferred)
(Tarkowski et al., 2010). The data from Tarkowski’s group indicate
that TE cells from an early mouse blastocyst are rarely pluripotent
(Suwinska et al. 2008; Tarkowski et al., 2010). TE cells from bovine
embryos are pluripotent at least until the expanding blastocyst stage
on Day 8 (Berg et al., 2011). We found that human outer cells
were able to recompact and develop into blastocysts with an ICM
up to the 64-cell stage, suggesting that their plasticity remains until a
later cell stage in the human as compared with the mouse but not
as long as in the bovine. Moreover, when the position of blastomeres
of early mouse blastocysts is randomly changed, these embryos
748
overcome the perturbation by sorting the cells back to their original
inside or outside position rather than by changing their developmental
direction (Suwinska et al., 2008). We found that human TE cells from a
full blastocyst do not sort themselves back to their outside position
when placed in an inside position. They change their developmental
direction, integrate within the ICM cells and some cells start to
express the pluripotency marker NANOG.
Our finding has important implications in reproductive medicine
because plasticity allows the early embryo to overcome perturbations
in its organization until the very late stages of preimplantation development. This implies that the impaired implantation capacity of embryos
after a 2-cell biopsy for PGD is linked to cell loss (De Vos et al., 2009).
It would also mean that in the clinic, blastocysts lacking an ICM may
benefit from manipulation (decavitation and decompaction) in order
to generate an inner cell population and, subsequently, an embryo
which is capable of implanting and developing further. Moreover,
this finding may have important implications in regenerative medicine
because full blastocyst TE cells could be a source of hESC lines, avoiding the destruction of the embryo which continues to develop normally in vitro and may be transferred into a uterus to obtain offspring
(Geens et al., 2009).
We conclude that full human blastocyst TE cells are not yet committed towards TE epithelium. Therefore, as they are a potential
source of hESC, the future development of hESC lines will provide sufficient material for the analysis of additional markers and demonstration of the ability of cells to differentiate into ectoderm, mesoderm
and endoderm: these data would definitively confirm our conclusion
that embryos reconstructed with TE cells are a source of pluripotent
cells.
Acknowledgements
We are grateful to the patients donating their embryos and gametes to
create embryos for research. We are also grateful to the colleagues of
the Centre for Reproductive Medicine, the Centre for Medical Genetics of the UZ Brussel, in particular Marleen Carlé for assisting with the
labeling experiments, and the Department of Reproduction and
Genetics of the VUB. C.P. and A.V. are Research Fellow at the Scientific Research Foundation—Flanders (FWO-Vlaanderen) and OZR.
Authors’ roles
H.V.d.V. contributed to conception and design, provision of study
material, experimental work, data analysis and interpretation and
writing of the manuscript; C.P. and G.C. contributed to experimental
work, data analysis and interpretation, revision and final approval of
the manuscript; A.V. contributed to experimental work and final approval of the manuscript; J.S. contributed to experimental work and
final approval of the manuscript; I.L. and H.T. contributed to revision
and final approval of the manuscript; P.D. contributed to final approval
of the manuscript.
Funding
Scientific Research Foundation—Flanders (FWO-Vlaanderen) and
Research Council (OZR) of the Vrije Universiteit Brussel.
De Paepe et al.
Conflict of interest
None declared.
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